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Abstract

Introduction

Several recent studies have shown that a positive fluid balance in critical illness
is associated with worse outcome. We tested the effects of moderate vs. high-volume
resuscitation strategies on mortality, systemic and regional blood flows, mitochondrial
respiration, and organ function in two experimental sepsis models.

Methods

48 pigs were randomized to continuous endotoxin infusion, fecal peritonitis, and a
control group (n = 16 each), and each group further to two different basal rates of
volume supply for 24 hours [moderate-volume (10 ml/kg/h, Ringer's lactate, n = 8);
high-volume (15 + 5 ml/kg/h, Ringer's lactate and hydroxyethyl starch (HES), n = 8)],
both supplemented by additional volume boli, as guided by urinary output, filling
pressures, and responses in stroke volume. Systemic and regional hemodynamics were
measured and tissue specimens taken for mitochondrial function assessment and histological
analysis.

Conclusions

High-volume resuscitation including HES in experimental peritonitis and endotoxemia
increased mortality despite better initial hemodynamic stability. This suggests that
the strategy of early fluid management influences outcome in sepsis. The high mortality
was not associated with reduced mitochondrial complex I- or II-dependent muscle and
hepatic respiration.

Introduction

Severe sepsis and septic shock are major causes of death in intensive care patients
[1,2]. Most deaths from septic shock can be attributed to either cardiovascular or multiorgan
failure [3]. The causes of organ dysfunction and failure are unclear, but inadequate tissue perfusion,
systemic inflammation, and direct metabolic changes at the cellular level are all
likely to contribute [4-6].

Fluid resuscitation is a major component of cardiovascular support in early sepsis.
Although the need for fluid resuscitation in sepsis is well established [7], the goals and components of this treatment are still a matter of debate. Several
recent studies have shown that a positive fluid balance in critical illness is strongly
associated with a higher severity of organ dysfunction and with worse outcome [8-14]. It is unclear whether this is the primary consequence of fluid therapy per se, or reflects the severity of illness.

We hypothesized that the fluid resuscitation strategy has an impact on sepsis-related
metabolic and cellular alterations, and outcome in sepsis. To test this hypothesis,
we used two different basal rates of volume supply (to mimic 'restrictive' and 'wet'
approaches), supplemented by additional volume boli, when clinically relevant and
commonly used physiological variables such as urinary output or filling pressures
decreased. We measured the effects of these two volume approaches on systemic and
regional blood flows, organ function and mortality. As no experimental model can directly
be extrapolated to clinical sepsis and the effects of fluid resuscitation may be model-dependent
[15,16], two different sepsis models - fecal peritonitis and endotoxemia - were studied.

Materials and methods

The study was performed in accordance with the National Institutes of Health guidelines
for the care and use of experimental animals and with the approval of the Animal Care
Committee of the Canton of Bern, Switzerland.

The experimental design included two factors: the model of sepsis (control, peritonitis,
endotoxemia) and the strategy of fluid resuscitation (moderate volume or high volume).
A full factorial design with six experimental groups was used.

Animal preparation and experimental setting

Pigs of both sexes (weight: median 41 kg; range 38 to 44 kg) were fasted overnight.
They were then premedicated, anesthetized with pentobarbital, intubated endotracheally
and ventilated (volume control mode; Servo ventilator 900 C; Siemens-Elema®, Solna, Sweden) with 5 cm H2O positive end-expiratory pressure. Anesthesia was maintained with pentobarbital (7
mg/kg/h) and fentanyl (25 μg/kg/h during operation and 3 μg/kg/h afterwards), and
pancuronium (1 mg/kg/h) was used for muscle relaxation. A single dose of 1.5 g cefuroxime
was injected before surgery. An esophageal Doppler probe (Deltex®, Chichester, UK) was inserted, and catheters for pressure measurement and blood sampling
were placed into the carotid, hepatic and pulmonary arteries, and into the jugular,
hepatic, portal, renal and mesenteric veins. Ultrasound Doppler flow probes (Transonic® System Inc., Ithaca, NY, USA) were positioned around the carotid, superior mesenteric,
splenic and hepatic arteries, and celiac trunk and portal vein. Laser Doppler needle
and surface probes (Optronics®, Oxford, UK) were inserted into the liver and kidney, and fixed on the surface of
gastric and jejunal mucosa and the kidney. More details on the surgical procedure
are described in the supplement [see Additional Data File 1].

Experimental protocol

After surgery, approximately 12 hours was allowed for hemodynamic stabilization. During
this period, Ringer's lactate at 10 ml/kg/h was infused to keep hemodynamic stability.
The animals were then randomized into six groups (eight pigs in each): control, fecal
peritonitis, or endotoxin, each with either high (15 ml/kg/hr Ringer's lactate and
5 ml/kg/hr hydroxyethyl starch (HES) 130/04, 6% (Voluven®, Fresenius, Stans, Switzerland)) or moderate volume fluid resuscitation (10 mL/kg/hr
Ringer's lactate).

In the peritonitis groups, 1 g per kg of autologous feces, dissolved in warmed glucose
solution, was instilled in the abdominal cavity. In the other groups, the same amount
of sterile glucose solution was instilled. The intraperitoneal drains were clamped
during the first six hours. In the endotoxin groups, endotoxin (lipopolysaccharide
from Escherichia coli 0111:B4, 20 mg/l in 5% dextrose; Sigma®, Steinheim, Germany) was infused into the right atrium. The effect of endotoxin was
judged by the magnitude of pulmonary artery pressure. Initially, endotoxin was infused
at 0.4 μg/kg/h until mean pulmonary arterial pressure reached 35 mmHg and the animals
became hypotensive. The endotoxin infusion was then stopped, and if arterial hypotension
persisted (mean arterial pressure below 60 mmHg), 50 ml of HES was administered. If
an arterial blood pressure of more than 55 mmHg could not be restored, boluses of
adrenaline (5 to 10 μg/bolus) were injected to prevent acute right heart failure and
death. Adrenaline was only used to treat hypotension within one hour of the onset
of pulmonary artery hypertension. If mean pulmonary pressure subsequently decreased
below 30 mmHg, the endotoxin infusion was restarted (0.1 μg/kg/h) and increased hourly
by 30%, if necessary, to maintain mean pulmonary artery pressure at 25 to 30 mmHg.
After eight hours of endotoxin infusion, the infusion rate was kept constant.

Throughout the experiment (including the postoperative stabilization period), the
volume status was evaluated clinically every hour, and if signs of hypovolemia became
evident (pulmonary artery occlusion pressure ≤ 5 mmHg or urinary output ≤ 0.5 mL/kg/hour),
additional 50 ml boluses of HES were given regardless of study group. Fluid boluses
were repeated under stroke volume monitoring with esophageal Doppler for as long as
the stroke volume was increased by 10% or more. For the validity of esophageal Doppler
with respect to cardiac output measurement by thermodilution see Dark and Singer [17]. To maintain the differences between high- and moderate-volume groups, maximal additional
volume was restricted to 100 ml per hour in all groups. Vasopressors were not used.
If necessary, 50% glucose solution was administered to maintain blood glucose of 3.5
to 6 mmol/l, and the standard infusion rate was adjusted to maintain unchanged basal
volume supply.

The quadriceps muscle was biopsied at baseline, after six hours, and at the end of
the experiment, and the liver was biopsied at the end of the experiment, for mitochondrial
function measurement [see Additional Data File 1].

The animals were followed until 24 hours after randomization or until death, if earlier.
After 24 hours, the animals were euthanized with an overdose of potassium chloride.
Blood sampling, histological analysis and interpretation of causes of mortality are
described in the online supplement [see Additional Data File 1].

Statistical analysis

The SPSS 13.0 software package (SPSS Inc.®, Chicago, IL, USA) was used for statistical analysis. Normal distribution was assessed
by the Kolmogorov-Smirnov test.

Survival proportions between the groups were analyzed with the log rank test, followed
by post-hoc log-rank tests for groups 'low volume' vs. 'high volume' and for groups 'endotoxemia'
vs. 'fecal peritonitis' vs. 'controls'. Differences between groups were assessed by
multivariate analysis of variance for repeated measures using one dependent variable,
two between-subject factors -- model (control, endotoxemia, peritonitis) and volume
(moderate, high) -- and one within-subject factor (time). Significant time-volume
and time-model interactions were considered as effects of volume resuscitation and
experimental model, respectively. If significant interactions occurred, analysis of
variance (ANOVA) for repeated measures was performed in the individual involved groups
to assess where changes occurred.

Fluid input and balance were compared with one-way ANOVA. The Tukey post-hoc test was performed to assess differences between the models. For hepatic mitochondrial
analysis, univariate analysis of variance was used. Significant effects of the fixed
factors model and volume were further analyzed post hoc with the independent t-test. For comparison of mitochondrial function between survivors
and non-survivors, an analysis of variance for repeated measures was used for muscle
mitochondria and an independent t-test for liver mitochondria. Statistical significance
was considered at P < 0.05. In post-hoc testing, the difference between groups with the lowest P value (even when >0.05) was considered responsible for the observed significant results
in primary testing. Data are expressed as mean ± standard deviation.

Results

Fluid balance

The three moderate-volume groups received an average of 11.0, and the high-volume
groups 2.4 boli of additional volume. The total fluid balance was markedly higher
in the high-volume groups (P < 0.001; Figure 1). Both peritonitis groups exhibited significantly higher fluid balances than their
matching other groups (P = 0.001).

Mortality

Eight animals had to be excluded from the analysis due to acute right-heart failure
and death within minutes after the start of endotoxin infusion (n = 7) and gut perforation
with rapid development of septic shock (n = 1). We found differences in mortality
(P < 0.001), with highest values in the peritonitis high-volume (n = 7; 88%) and endotoxin
high-volume (n = 6, 75%) groups. Mortality was higher in high- vs. low-volume groups,
and in septic vs. control groups (P < 0.01, both), but did not differ between endotoxemia and fecal peritonitis groups.
The respective median survival times were 17.5 and 16 hours. Mortality was 50% (n
= 4) in the peritonitis moderate-volume group and 12.5% (n = 1) in the endotoxin moderate-volume
group, with median survival times of 23.5 and 24 hours, respectively. One animal in
the control high-volume group died at 23.5 hours, while all moderate-volume control
pigs survived until the end of the experiment (Figure 2).

Figure 2. Survival curves of all experimental groups. log rank test: P < 0.001. The cause of death is also shown for each pig.

Lungs

The oxygenation index (partial pressure of arterial oxygen to fraction of inspired
oxygen) decreased in all groups over the course of the experiment, but most in the
peritonitis groups (P = 0.001; Table 3). The respiratory plateau pressure increased in all groups, with the highest values
in control and peritonitis high-volume animals (P = 0.04; Table 3). The dynamic compliance of the respiratory system decreased in all groups, without
differences related to volume or model. Lung histology revealed the presence of colloid
plaques and atelectases in all groups of animals [see Figures S4 and S5 in Additional
Data File 3]. Colloid plaques tended to be more frequently present in the high-volume groups
(84%) in comparison with their respective moderate-volume groups (59%). Atelectases
were present in 50% or more of the animals of all groups.

Histology revealed severe damage in five of six endotoxin high-volume animals (83%)
and in 30% to 40% of the animals in the endotoxin and peritonitis moderate-volume
groups (Figure 3). Storage of starch (HES) in the tissues was detectable as a purple fluid in H&E-stained
tissue sections, as confirmed by positive Periodic acid-Schiff staining. This fluid
was mainly found in dilated tubules. There was no predilection for one of the groups
(Figure 4).

Heart

Discussion

The main finding of this study was that high-volume fluid resuscitation including
HES increased mortality in sepsis. The increased mortality was observed in both models
of fecal peritonitis and endotoxemia. Both these established large-animal sepsis models
share many of the features of clinical sepsis, including hypovolemia if untreated,
normo- or hyperdynamic circulation with volume resuscitation, high mortality, and
signs of progressive organ dysfunction despite cardiovascular and respiratory support.

Despite major differences in volume supply, differences in hemodynamic responses between
the groups were either modest or appeared late: the most prominent difference was
progressive pulmonary artery hypertension and increased cardiac filling pressures
in the high-volume groups, especially in peritonitis. We did not perform echocardiography,
so direct evaluation of myocardial function was not possible. In particular the severity
of right ventricular dysfunction may have been underestimated. The increased cardiac
enzymes in all high-volume groups support the concept that relevant myocardial damage
occurred. Fluid loading in septic animals has been shown to induce a large reduction
in vascular tone, which could be attenuated by inhibition of nitric oxide synthesis
[18]. It is conceivable to argue that high amounts of volume can promote vascular leak
and interstitial edema in septic states by releasing nitric oxide and/or other vasodilating
agents. This effect would be even more exaggerated when filling pressures increase
as an effect of cardiac dysfunction. In our study, lung dysfunction, reflected in
impaired oxygenation index and mechanics, was the cause of approximately every third
death in the high-volume septic groups and none in the moderate-volume groups. Renal
perfusion was also predominantly affected in the high-volume septic animals; especially
in peritonitis, despite high cardiac output and relatively well-preserved mean arterial
pressure.

The criteria for and targets of fluid management in sepsis are controversial. In clinical
sepsis, recent guidelines - based mainly on expert opinions (Surviving Sepsis Campaign)
- have recommended fluid administration to restore cardiac filling pressures to at
least 12 mmHg during mechanical ventilation [19]. In mechanically ventilated patients or patients with known pre-existing decreased
ventricular compliance, central venous pressure targets of 12 to 15 mmHg have been
suggested [20]. In clinical sepsis trials where fluid was administered to optimize hemodynamics,
central venous pressures of up to 22 mmHg have been reached [21]. In the present study, only the high-volume groups reached levels recommended by
the Surviving Sepsis campaign, with the high-volume peritonitis group exceeding these
levels, and these were also the groups with the highest mortality rates. Although
our approach of two different basal rates of volume supply can be criticized, it should
be noted that even animals in the high-volume groups received additional fluid boluses
as a result of the appearance of clinical signs of hypovolemia. In clinical sepsis
trials, the total amount of fluid given is rarely indicated. It is evident that high
targets for filling pressures will result in large amounts of administered fluids
when capillary leakage is present, and the administered fluid does not translate into
a significant increase in venous return. For example, in the study by Rivers and colleagues
[7], patients received a mean (± standard deviation) of 5 (± 3) liters of fluid within
the first six hours. In other patient groups, including patients with multiorgan failure
and sepsis, patients received 13 to 30 liters of fluid for resuscitation within 24
hours [22,23]. There is growing evidence that large amounts of fluids may be harmful, especially
in septic patients [11,24,25], but also in other patient groups [22]. Our results point in the same direction.

Many of the experimental sepsis studies, including the present one, have used substantially
larger doses of HES than is recommended in the clinical setting. Recent trials in
clinical sepsis have found a dose-related association between HES and renal failure
in sepsis [26]. Although a different HES solution was used in the present study, we cannot exclude
that HES influenced the outcomes due to its pharmacological properties. Nevertheless,
urinary output increased and creatinine concentrations decreased in both control and
endotoxin high-volume groups. Furthermore, histology revealed major abnormalities
in the endotoxin high-volume group but not in the peritonitis high-volume group.

Mitochondrial dysfunction has been suspected to contribute to mortality in sepsis.
We found that neither the models of sepsis nor the volume resuscitation strategy resulted
in altered hepatic or muscle mitochondrial complex I- and II-dependent respiration.
We cannot exclude sepsis-induced impairment of mitochondrial function by mechanisms
not tracked by our methods [27-29]. Nevertheless, normal arterial lactate concentrations and hepatic vein lactate/pyruvate
ratios in all groups do not seem to suggest major mitochondrial respiration abnormality
either. Recently, energetic failure of peripheral blood mononuclear cells in sepsis
has been implicated in the modulation of immune response [30]. Nevertheless, how volume overload potentially aggravates early immune suppression
remains unclear.

The relevance of our results for clinical sepsis deserves consideration. Although
both sepsis models have many similarities with clinical sepsis, there are important
differences, both in the models per se and in the treatments tested. First, both models included major abdominal surgery
before induction of sepsis. The impact of recent surgery on metabolic demands and
blood flow will inevitably be superimposed on the effects of sepsis. Second, the volume
support was started at the same time that sepsis was induced, whereas clinical sepsis
is typically associated with a delay in starting the treatment. Third, early antibiotics
improve the outcome of clinical sepsis, but this was not included in our treatment.
Fourth, hypotension not responsive to fluids alone is treated with vasoactive agents
in clinical sepsis. As we did not use any inotropes or vasopressors, this clearly
limits the extrapolation of our results to clinical sepsis.

Conclusions

We conclude that aggressive volume resuscitation initially maintains systemic hemodynamics
and regional blood flow in experimental endotoxemia and fecal peritonitis. However,
it markedly increases mortality. Supplemental fluids should be used only as long as
tissue perfusion can be improved. Future experiments should more closely mimic the
natural course and treatment of sepsis.

Abbreviations

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SMJ and JT designed the study, supervised the experiments, and revised the manuscript.
SB, HB, FP, VK, JG, VK, and LBH conducted the experiments, including anesthesia. SB
drafted the manuscript. TR performed the statistical analysis. TR, FP, SD, and EB
performed the mitochondrial experiments. SD and UK performed the remaining laboratory
analyses. LEB and GB performed surgery and revised the manuscript. PL supervised all
laboratory analysis and revised the manuscript. LW performed all histological analyses.
All authors read and approved the final manuscript.

Acknowledgements

This research was supported by grant 3200BO/102268, made available by the Swiss National
Fund, Bern, Switzerland. We thank Ms. Colette Boillat and Ms. Alice Zosso (Department
of Pediatric Surgery, Inselspital, Bern University Hospital and University of Bern)
for technical assistance, especially regarding histology, and Ms. Jeannie Wurz (Department
of Intensive Care Medicine) for editing the manuscript.

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